The overall goal of the research is to characterize the solution structure and dynamics of a fifteen residue peptide (derived from HIV-1 gpl2O protein) when it is conjugated to the carrier protein ovalbumin or bound to the antigen binding fragment (Fab) of two antibodies-5023 and 5025A. Both antibodies were raised against the peptide-ovalbumin conjugate. Parallel studies of this peptide when conjugated to a second, model carrier protein, basic pancreatic trypsin inhibitor (BPTI), and complexed with the Fab of a third antibody (to be raised against this BPTI-peptide conjugate) will also be performed. One- and two-dimensional solution NMR methods will be used to investigate the structure and dynamics of the peptide moiety of these complexes and conjugates. Information derived from the conjugated peptide studies should address questions regarding antipeptide antibody induction and the role of the carrier protein during induction. Studies of the Fab-peptide complexes will benefit the understanding of antigen recognition by antibodies in general. This research should contribute to the anti-peptide antibody knowledge base overall and lead to improvements in the use of these antibodies as diagnostic reagents or in the development of vaccines. Conventional two-dimensional COSY, TOCSY and NOESY experiments will be used to assign proton resonances of the BPTI-coupled peptide. NOESY measurements conducted upon the BPTI-coupled peptide will provide peptide interproton distance estimates which, for a constrained, conjugated peptide, can be used for distance geometry calculations to determine the three-dimensional structure of the peptide. BPTI-coupled peptide dynamics will be investigated via reverse-detection techniques and relaxation measurements. The Fab complexes and ovalbumin conjugate will be investigated using isotope-edited NMR methods. Synthetic peptides with 15- N, 13-C and 2 H stable isotopes incorporated at specific residues will be complexed with Fabs or conjugated to ovalbumin. Isotope-edited relaxation measurements which are feasibly conducted upon the Fab complexes and ovalbumin-coupled peptide will enable a comparison between the relaxation properties of the conjugated and complexed forms of the peptide to detect differences in terms of dynamics between the two. Peptide proton internuclear distance estimates win be derived from isotope-edited NOE measurements conducted upon the Fab complexes and ovalbumin conjugate composed of native (undeuterated) Fab and ovalbumin, respectively. For the Fab complex, "refinement" of these initial estimates may be necessary, due to potential indirect magnetization transfer between peptide and Fab; this involves conducting NOE measurements upon complexes containing specifically deuterated antibody and/or peptide. In either case, distance estimates obtained from the Fab-peptide and ovalbumin conjugate NOE studies provide information regarding the overall peptide proton geometry which will be used to structurally characterize the Fab-associated and ovalbumin-coupled peptide forms, respectively. Further comparisons between the structures of the peptide bound to the 5023 and 5025 Fabs will be made to understand the physical basis for the specificity differences between these two antibodies.